假基因鉴定及其功能分析

被称为"垃圾基因"的假基因是真核生物基因组中的重要组成部分。近年来对假基因的功能研究表明其并非是基因组中的沉默成员。如一些假基因参与RNA转录,一些假基因转录本能够形成小干扰RNA(siRNA),通过小RNA干扰作用调节功能基因。另外,还有研究发现,一些假基因能够通过microRNA调节肿瘤抑制因子。然而,对假基因功能的深入挖掘需要建立在对其更精准、更全面的鉴定基础之上。随着各物种全基因组测序的完成及序列比对算法的完善,全面而又精确地鉴定假基因已经成为可能。下文就近年来假基因相关鉴定方法、调节功能以及在进化上的意义进行了阐述,并对未来假基因研究方向进行了展望。     

人类基因组上的假基因

假基因是基因组上与编码基因序列非常相似的非功能性基因组DNA拷贝,一般情况都不被转录,且没有明确生理意义。假基因根据其来源可分为复制假基因和已加工假基因。迄今为止,明确鉴定的人类假基因多为已加工假基因,有8000个之多。在Swiss-Prot/TrEMBL收录的编码蛋白质的将近25500个基因序列中,约10％在基因组中有一个或多个近全长已加工假基因。其余的功能基因都没有已加工假基因。核糖体蛋白基因具有最多数量的已加工假基因,约有l700个(占已加工假基因数的22％),少数基因,如cyclophilinA、肌动蛋白(actin)、角蛋白(keratin)、GAPDH、细胞色素C(cytochromec)和nucleophosmin等则有很多份已加工假基因。总体上讲,假基因在人类染色体上的分布与染色体长度成比例,但已加工假基因在GC含量为41％～46％的染色体区域密度最高。已加工假基因的拷贝数和功能基因在生殖器官中的表达高度一致,说明许多假基因发生在胚胎阶段,另外也和基因中GC含量和基因大小密切相关。假基因的准确鉴定对基因组进化、分子医学研究和医学应用具有重要意义。     

黒腹果蝇中嵌合新基因的进化命运和表达模式

新基因的起源和进化对基因组多样性的产生具有重要的贡献.新基因起源常常通过外显子重排而形成嵌合的基因结构,以产生具有新功能的蛋白质.该文调查了在黒腹果蝇中的14个新起源的嵌合基因在群体中的多态性,发现其中8个在群体中的核苷酸多态性会引起提前终止子,而其他6个在群体中编码框都完整且其中4个受到负选择.研究结果表明,嵌合新基因起源后可能存在两种命运:积累提前终止子突变而假基因化,或者表现出一定功能而受自然选择固定下来.基因表达的数据显示,与RNA介导外显子重排(逆转座)形成的新基因不一样,这些由DNA水平外显子重排产生的新基因没有精巢或者雄性特异性表达模式,而是表现出更为多样性的时空表达模式,这提示尽管通过DNA水平外显子重排产生的新基因可能正在变成假基因或者非蛋白质编码的RNA基因,但它们依然可能具有进化出广泛的生物学功能的潜力.     

假基因研究进展

假基因是功能基因的缺陷拷贝,它在序列结构上与功能基因非常相似,但已丧失了正常的蛋白质编码功能．假基因曾被认为是一类典型的非编码“垃圾DNA”,而如今人们发现假基因在基因表达调控和基因组进化过程中发挥着重要作用．从假基因的起源、序列结构特征、假基因的识别、假基因在染色体上的分布、分子进化规律,以及假基因功能等几个方面较为全面地介绍了该领域的最新研究进展．     

假基因的组成、分布及其分子进化

假基因(pseudogene)是指基因组中与正常基因序列相似, 但是缺乏功能的DNA 序列。通过序列同源性搜索, 可以收集基因组中假基因的群体特性、染色体分布和同源家族等特性。假基因很好地保留了数百万年前基因组中祖先基因的分子记录, 被视为“基因化石”, 因此假基因在进化和比较基因组学中是重要的资源。应用假基因和基因比较体系, 可以探究生物基因的进化史和基因组稳定性。如: 用Ka/Ks比值确定假基因的自然选择压、物种亲缘关系和进化距离, 分析假基因自身的进化趋势, 探讨DNA 突变的成因等。     

RT-PCR检测HUTP14A mRNA时排除假基因HUTP14C干扰的方法

人类U3蛋白14C基因(HUTP14C)是人类U3蛋白14A基因(HUTP14A)的假基因。两者转录本序列同源性高达95%。常规RT-qPCR技术在检测HUTP14A mRNA丰度时,HUTP14C的存在会影响检测结果。本研究旨在建立检测HUTP14A mRNA时排除HUTP14C干扰的RT PCR方法。本研究设计出能分别从多种肿瘤细胞DNA和RNA中特异性扩增HUTP14A和HUTP14C的引物,避免假基因HUTP14C对其同源基因HUTP14A检测的干扰。在检测细胞系HUTP14A mRNA时,通过DNaseⅠ消除RNA中污染的HUTP14C DNA,用靶向HUTP14C 3′-UTR的siRNA沉默HUTP14C mRNA后,再用RT PCR检测HUTP14A mRNA丰度,使结果更加准确。在18对肝癌及癌旁组织中,利用特异性引物进行RT PCR检测,HUTP14A和HUTP14C mRNA的表达略高于癌旁组织。本研究提示,针对有假基因存在的功能基因,对其mRNA丰度进行检测时,在提取细胞或组织总RNA后,用DNaseⅠ处理,再用RNA直接进行PCR扩增,排除DNA污染后,再进行RT-PCR或RT-qPCR扩增。大多假基因具有较长的3′-UTR区,在该区域设计siRNA特异性沉默假基因的mRNA后,用RT-qPCR检测功能基因的mRNA丰度,可以排除假基因mRNA的影响。在病理组织中检测功能基因的mRNA丰度时,可以根据假基因和其功能基因的序列差异设计出特异扩增功能基因的引物,从假基因的3′-UTR区设计特异扩增假基因的引物,通过RT-qPCR技术分别检测二者的mRNA。     

假基因的组成、分布及其分子进化

假基因（pseudogene）是指基因组中与正常基因序列相似,但是缺乏功能的DNA序列.通过序列同源性搜索,可以收集基因组中假基因的群体特性、染色体分布和同源家族等特性.假基因很好地保留了数百万年前基因组中祖先基因的分子记录,被视为＂基因化石＂,因此假基因在进化和比较基因组学中是重要的资源.应用假基因和基因比较体系,可以探究生物基因的进化史和基因组稳定性.如：用Ka/Ks比值确定假基因的自然选择压、物种亲缘关系和进化距离,分析假基因自身的进化趋势,探讨DNA突变的成因等.     

假基因的发现与研究

随着人类基因组计划的顺利实施,人们分离、鉴定新基因的速度越来越快,对于占人类基因组97%的非表达序列的研究,即对所谓"垃圾"DNA的研究已成为全球范围内关注的热点.现就假基因的发现、命名和分类、特性和分布、产生、作用机理、功能、进化及展望等方面进行论述.     

基于DNA和RNA的双功能Semliki森林病毒复制子载体的构建

以semliki森林病毒衍生的复制子载体pSFV1和辅助载体pSFV-helper2为骨架, 用CMV IE和T7启动子替换SP6启动子并在3′ UTR下游插入BGH转录终止子,构建了基于DNA和RNA的复制子表达载体pSMCTA和辅助载体pSHCTA。在DNA和RNA二种递送方式上证实该表达载体可高水平表达外源基因,与辅助载体共转染可制备具有感染能力并能表达外源基因的重组病毒颗粒。构建的基于DNA和RNA的双功能复制子载体显著地提高SFV载体应用范围,在体外可用于高水平表达外源基因及大规模制备重组病毒颗粒,在体内也可用于研制复制子疫苗和基因治疗载体。     

基因组常用术语解释(下)

蛋白组 (Proteome) :一个特定基因组产生的全部蛋白质 (The full complement of proteins produced by aparticular genome)。蛋白组学 (Proteomics) :研究基因组编码的全部蛋白质的科学 (The study of the full set of proteins en-coded by a genome)。假基因 (Pseudogene) :类似于基因但却没有功能的 DNA序列 ,或许是曾经具有功能的基因积累突变后残留所致 (A sequence of DNA similar to a gene butnonfunctional;probably the remnantof a once- func-tional gene that accumulated mutations)。调控区 (Regulatory region) :控…     

High-level expression of pseudogenes in Mycobacterium leprae

Recent studies have revealed that some RNAs are transcribed from noncoding DNA regions, including pseudogenes, and are functional as riboregulators. We have attempted to assess the gene expression profile throughout the Mycobacterium leprae genome using an array technique. Twelve highly expressed gene regions were identified that show an alteration in expression levels upon infection. Six of these were pseudogenes. Although M. leprae has an exceptional number and proportion of pseudogenes among species, our results suggest that some of the M. leprae pseudogenes are not just 'decayed' genes, but may have a functional role.     

The small RNA content of human sperm reveals pseudogene-derived piRNAs complementary to protein-coding genes

At the end of mammalian sperm development, sperm cells expel most of their cytoplasm and dispose of the majority of their RNA. Yet, hundreds of RNA molecules remain in mature sperm. The biological significance of the vast majority of these molecules is unclear. To better understand the processes that generate sperm small RNAs and what roles they may have, we sequenced and characterized the small RNA content of sperm samples from two human fertile individuals. We detected 182 microRNAs, some of which are highly abundant. The most abundant microRNA in sperm is miR-1246 with predicted targets among sperm-specific genes. The most abundant class of small noncoding RNAs in sperm are PIWI-interacting RNAs (piRNAs). Surprisingly, we found that human sperm cells contain piRNAs processed from pseudogenes. Clusters of piRNAs from human testes contain pseudogenes transcribed in the antisense strand and processed into small RNAs. Several human protein-coding genes contain antisense predicted targets of pseudogene-derived piRNAs in the male germline and these piRNAs are still found in mature sperm. Our study provides the most extensive data set and annotation of human sperm small RNAs to date and is a resource for further functional studies on the roles of sperm small RNAs. In addition, we propose that some of the pseudogene-derived human piRNAs may regulate expression of their parent gene in the male germline.     

Structural and functional analysis of the goat epsilon-globin genes

Since none of the vertebrate beta-globin loci studied to date has more than two functional embryonic beta-like globin genes, it would be unique if all six goat embryonic beta-globin genes were required for its survival. In this study we have asked whether all six embryonic genes in the goat are functional. This question has been addressed by examining the transient expression of these genes in HeLa cells and correlating these results with the sequence information obtained to date. Our studies show that only epsilon I and epsilon II are functional while the remaining four epsilon-globin genes are nonfunctional, i.e., pseudogenes. Interestingly, the two active epsilon-globin genes are located at the 5' end of the locus. While this unusual inactivation pattern may be the result of chance, it could also have resulted because the two duplication events, of the ancestral gene set epsilon-epsilon-psi beta-beta, did not include distally located regulatory region(s) essential for epsilon-globin gene expression. Once separated from the 5'-regulatory sequences the remaining four embryonic genes (epsilon III, epsilon IV, epsilon V and epsilon VI) accumulated mutations and became pseudogenes.     

Pseudogenes: Pseudo or Real Functional Elements?

Pseudogenes are genomic remnants of ancient protein-coding genes which have lost their coding potentials through evolution. Although broadly existed, pseudogenes used to be considered as junk or relics of genomes which have not drawn enough attentions of biologists until recent years. With the broad applications of high-throughput experimental techniques, growing lines of evidence have strongly suggested that some pseudogenes possess special functions, including regulating parental gene expression and participating in the regulation of many biological processes. In this review, we summarize some basic features of pseudogenes and their functions in regulating development and diseases. All of these observations indicate that pseudogenes are not purely dead fossils of genomes, but warrant further exploration in their distribution, expression regulation and functions. A new nomenclature is desirable for the currently called ‘pseudogenes’ to better describe their functions.